There has been increasing interest in the relationship between rapid effects of steroids and steroid-plasma membrane interaction. This laboratory has previously reported that progesterone increases human sperm cytosolic free calcium ([Ca2+]i) and thereby initiates the human sperm acrosome reaction (AR) in less than 1 min. Herein, to test whether progesterone acts at the sperm plasma membrane, progesterone 3-(O-carboxymethyl)oxime: bovine serum albumin (BSA) conjugate (free of unconjugated progesterone) was added to capacitated human sperm. Fura-2 assays were used to detect less than 1 min changes in [Ca2+]i, and indirect immunofluorescence was used to assay the AR occurring 1 min after stimulus addition. The conjugate increased [Ca2+]i and the AR (though less than did unconjugated progesterone). Enzyme immunoassays demonstrated that the concentrations of unconjugated progesterone in conjugate-treated sperm suspensions did not increase over those of control suspensions. Since the progesterone: BSA conjugate presumably does not cross the sperm plasma membrane, progesterone must act at that membrane to increase [Ca2+]i and the AR.